Antimicrobial flush solutions

ABSTRACT

The present invention provides antimicrobial solutions that comprise at least one alcohol, at least one antimicrobial agent and at least one chelator and/or anticoagulant. Also provided are methods for rapidly reducing a microbe or a virus from surfaces including surfaces of indwelling medical devices and organic surfaces such as skin and sutures, and inorganic surfaces such as hospital equipment, pipelines etc.

The present application claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 60/476,555, filed on Jun. 6,2003. The entire text of the above-referenced disclosure is specificallyincorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medicine andmicrobiology. More particularly, it concerns methods of reducingmicrobial organisms from indwelling medical devices, medical equipmentand other surfaces.

2. Description of Related Art

Medical devices, such as vascular catheters, have improved the qualityof medical care. However, infections resulting from the colonization oforganisms embedded in biofilm are the most frequent complicationassociated with the use of these and other indwelling and/or prostheticdevices. In fact, infections are the most serious complicationsassociated with indwelling central venous catheters (CVCs) (Maki et al.,1998). It is estimated that more than 200,000 catheter-relatedbloodstream infections (CRBSI) occur annually in the United States alone(Kluger et al., 1999). Staphylococcus epidermidis, Staphylococcus aureusand Candida species are the leading organisms causing CRBSI (Maki etal., 1998; Raad et al., 2002).

Because intralumenal colonization is the major source for the migrationof organisms leading to bloodstream infections in long-term siliconecatheters (Raad et al., 1993), recent guidelines by the CDC andInfectious Diseases Society of America have proposed the use ofintralumenal antimicrobial lock solutions for the prevention andtreatment of CRBSI (Mermel et al., 2001; Centers for Disease Control andPrevention, 2002). Most long-term CVCs are typically flushed withheparin. An antimicrobial/anticoagulant combination consisting ofvancomycin/heparin with and without ciprofloxacin was shown to reducethe risk of catheter-related bacteremia caused by gram-positiveorganisms (Carratala et al., 1999; Henrickson et al., 2002; Schwartz etal., 1990). However, with the rise of incidences of infection byvancomycin resistant gram-positive bacteria, concerns have been raisedover the use of vancomycin flush solutions and their potential forincreasing the risk of vancomycin resistance (Spafford et al., 1994).

Recently the present inventor demonstrated that a flush solutioncomprising minocycline and EDTA (M-EDTA) is highly efficacious inpreventing catheter-related colonization, bacteremia and endocarditis inrabbits (Raad et al., 2002). When compared to a heparin flush solution,M-EDTA was found to decrease the risk of catheter-related colonizationand infection in hemodialysis patients as well as pediatric cancerpatients (Bleyer et al., 2000; Chatzinikolaou et al., 2002). EDTA has anequivalent anticoagulant activity to heparin (Reardon et al., 1991). Ananticoagulant in flush solutions is necessary to prevent the thromboticocclusion of the catheter lumen.

Although M-EDTA has been found to be efficacious in preventing CRBSI,this solution may not be applicable given some of the limitations of thereal world of clinical practice. In the animal and clinical studies, theM-EDTA lock solution was required to be exposed to the surface of theindwelling medical device, such as the lumen of catheters, for at least4 hours. In vitro studies have also shown that M-EDTA requires at least4 hours of dwell time to eradicate organisms that colonize the lumen ofthe catheter (see in particular data in U.S. Pat. No. 5,362,754, columns11 and 12, and Tables 3, 4 and 5 as well as in U.S. Pat. No. 5,688,516,columns 15 and 16, and Tables 3, 4, and 5). Providing a four hourexposure time to reduce microbes using the M-EDTA solution is usuallynot possible in critically ill patients who require continuous infusiontherapy, including parenteral nutrition.

Thus, there is an acute need in the art to develop compositions andmethods for rapid reduction and/or eradication of microbes fromindwelling medical devices without interruption of the use of the devicein patients for too long a period. In addition, there is also a need forbetter and improved antimicrobial compositions.

SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations in the artand provides compositions that reduce or eradicate microbial agents fromsurfaces wherein the compositions comprise at least one antimicrobialagent, at least one chelator and/or anticoagulant, and at least onealcohol. The present invention also provides methods to rapidly reduceor eradicate microbial agents from surfaces.

Therefore, provided are antimicrobial solutions comprising at least onealcohol, at least one antimicrobial agent and at least one chelatorand/or anticoagulant. “Antimicrobial agents” that are comprised in thesolutions of the present invention include antibacterial agents,antifungal agents, antiviral agents as well as antiseptic agents. Thesecomponents are present in effective amounts to reduce microbial growth.

In some embodiments of the invention, the antimicrobial agent is anantibacterial agent. While any antibacterial agent may be used in thepreparation of the instant antimicrobial solutions, some non-limitingexemplary antibacterial agent(s) include those classified asaminoglycosides, beta lactams, quinolones or fluoroquinolones,macrolides, sulfonamides, sulfamethaxozoles, tetracyclines,streptogramins, oxazolidinones (such as linezolid), clindamycins,lincomycins, rifamycins, glycopeptides, polymxins, lipo-peptideantibiotics, as well as pharmacologically acceptable sodium salts,pharmacologically acceptable calcium salts, pharmacologically acceptablepotassium salts, lipid formulations, derivatives and/or analogs of theabove.

Each of these classes of antibacterial agents have different mechanismsof action and are represented by several antibiotics a discussion ofwhich is presented below. However, the skilled artisan will recognizethat the invention is in no way limited to the agents set forth here andthat these agents are described merely as examples.

The aminoglycosides are bactericidal antibiotics that bind to the 30Sribosome and inhibit bacterial protein synthesis. They are typicallyactive against aerobic gram-negative bacilli and staphylococci.Exemplary aminoglycosides that may be used in some specific aspects ofthe invention include amikacin, kanamycin, gentamicin, tobramycin, ornetilmicin.

Beta lactams are a class of antibacterials that inhibit bacterial cellwall synthesis. A majority of the clinically useful beta-lactams belongto either the penicillin group (penam) or cephalosporin (cephem) groups.The beta-lactams also include the carbapenems (e.g., imipenem), andmonobactams (e.g., aztreonam). Inhibitors of beta-lactamase such asclavulanic acid and its derivatives are also included in this category.

Non-limiting examples of the penicillin group of antibiotics that may beused in the solutions of the present invention include amoxicillin,ampicillin, benzathine penicillin G, carbenicillin, cloxacillin,dicloxacillin, piperacillin, or ticarcillin, etc. Examples ofcephalosporins include ceftiofur, ceftiofur sodium, cefazolin, cefaclor,ceftibuten, ceftizoxime, cefoperazone, cefuroxime, cefprozil,ceftazidime, cefotaxime, cefadroxil, cephalexin, cefamandole, cefepime,cefdinir, cefriaxone, cefixime, cefpodoximeproxetil, cephapirin,cefoxitin, cefotetan etc. Other examples of beta lactams include mipenemor meropenem which are extremely active parenteral antibiotics with aspectrum against almost all gram-positive and gram-negative organisms,both aerobic and anaerobic and to which Enterococci, B. fragilis, and P.aeruginosa are particularly susceptible.

Examples of beta lactamase inhibitors include clavulanate, sulbactam, ortazobactam. In some aspects of the present invention, the antibacterialsolutions may comprise a combination of at least one beta lactam and atleast one beta lactamase inhibitor.

Macrolide antibiotics are another class of bacteriostatic agents thatbind to the 50S subunit of ribosomes and inhibit bacterial proteinsynthesis. These drugs are active against aerobic and anaerobicgram-positive cocci, with the exception of enterococci, and againstgram-negative anaerobes. Exemplary macrolides include erythromycin,azithromycin, clarithromycin.

Quinolones and fluoroquinolones typically function by their ability toinhibit the activity of DNA gyrase. Examples include nalidixic acid,cinoxacin, trovafloxacin, ofloxacin, levofloxacin, grepafloxacin,trovafloxacin, sparfloxacin, norfloxacin, ciprofloxacin, moxifloxacinand gatifloxacin.

Sulphonamides are synthetic bacteriostatic antibiotics with a widespectrum against most gram-positive and many gram-negative organisms.These drugs inhibit multiplication of bacteria by acting as competitiveinhibitors of p-aminobenzoic acid in the folic acid metabolism cycle.Examples include mafenide, sulfisoxazole, sulfamethoxazole, andsulfadiazine.

The tetracycline group of antibiotics include tetracycline derivativessuch as tigecycline which is an investigational new drug (IND),minocycline, doxycycline or demeclocycline and analogs such asanhydrotetracycline, chlorotetracycline, or epioxytetracycline. Thepresent inventors have previously shown that minocycline has a higherpenetration of the microbial biofilm layer than vancomycin and that EDTAis unique in effectively preventing and dissolving polysaccharide-richmicrobial glycocalyx (U.S. Pat. No. 5,362,754).

The streptogramin class of antibacterial agents is exemplified byquinupristin, dalfopristin or the combination of two streptogramins.

Drugs of the rifamycin class typically inhibit DNA-dependent RNApolymerase, leading to suppression of RNA synthesis and have a verybroad spectrum of activity against most gram-positive and gram-negativebacteria including Pseudomonas aeruginosa and Mycobacterium species. Anexemplary rifamycin is rifampicin.

Other antibacterial drugs are glycopeptides such as vancomycin,teicoplanin and derivatives thereof. Yet other antibacterial drugs arethe polymyxins which are exemplified by colistin.

In addition to these several other antibacterial agents such asprestinomycin, chloramphenicol, trimethoprim, fusidic acid,metronidazole, bacitracin, spectinomycin, nitrofurantion, daptomycin orother leptopeptides, oritavancin, dalbavancin, ramoplamin, ketolide etc.may be used in preparing the compositions described herein. Of these,metronidazole is active only against protozoa, such as Giardia lamblia,Entamoeba histolytica and Trichomonas vaginalis, and strictly anaerobicbacteria. Spectinomycin, is a bacteriostatic antibiotic that binds tothe 30S subunit of the ribosome, thus inhibiting bacterial proteinsynthesis and nitrofurantoin is used orally for the treatment orprophylaxis of UTI as it is active against Escherichia coli,Klebsiella-Enterobacter species, staphylococci, and enterococci.

In other embodiments, the antimicrobial agent is an antifungal agent.Some exemplary classes of antifungal agents include imidazoles ortriazoles such as clotrimazole, miconazole, ketoconazole, econazole,butoconazole, omoconazole, oxiconazole, terconazole, itraconazole,fluconazole, voriconazole (UK 109,496), posaconazole, ravuconazole orflutrimazole; the polyene antifungals such as amphotericin B, liposomalamphoterecin B, natamycin, nystatin and nystatin lipid formualtions; thecell wall active cyclic lipopeptide antifungals, including theechinocandins such as caspofungin, micafungin, anidulfungin, cilofungin;LY121019; LY303366; the allylamine group of antifungals such asterbinafine. Yet other non-limiting examples of antifungal agentsinclude naftifine, tolnaftate, mediocidin, candicidin, trichomycin,hamycin, aurefungin, ascosin, ayfattin, azacolutin, trichomycin,levorin, heptamycin, candimycin, griseofulvin, BF-796, MTCH 24,BTG-137586, pradimicins (MNS 18184), benanomicin; ambisome; nikkomycinZ; flucytosine, or perimycin.

In still other embodiments of the invention, the antimicrobial agent isan antiviral agent. Non-limiting examples of antiviral agents includecidofovir, amantadine, rimantadine, acyclovir, gancyclovir, pencyclovir,famciclovir, foscamet, ribavirin, or valcyclovir. In some embodimentsthe antimicrobial agent is an innate immune peptide or proteins. Someexemplary classes of innate peptides or proteins are transferrins,lactoferrins, defensins, phospholipases, lysozyme, cathelicidins,serprocidins, bacteriocidal permeability increasing proteins,amphipathic alpha helical peptides, and other synthetic antimicrobialproteins.

In other embodiments of the invention, the antimicrobial agent is anantiseptic agent. Several antiseptic agents are known in the art andthese include a taurinamide derivative, a phenol, a quaternary ammoniumsurfactant, a chlorine-containing agent, a quinaldinium, a lactone, adye, a thiosemicarbazone, a quinone, a carbamate, urea, salicylamide,carbanilide, a guanide, an amidine, an imidazoline biocide, acetic acid,benzoic acid, sorbic acid, propionic acid, boric acid, dehydroaceticacid, sulfurous acid, vanillic acid, esters of p-hydroxybenzoic acid,isopropanol, propylene glycol, benzyl alcohol, chlorobutanol,phenylethyl alcohol, 2-bromo-2-nitropropan-1,3-diol, formaldehyde,glutaraldehyde, calcium hypochlorite, potassium hypochlorite, sodiumhypochlorite, iodine (in various solvents), povidone-iodine,hexamethylenetetramine, noxythiolin, 1-(3-choroallyl)-3,5,7-triazo1-azoniaadamantane chloride, taurolidine, taurultam,N(5-nitro-2-furfurylidene)-1-amino-hydantoin, 5-nitro-2-furaldehydesemicarbazone, 3,4,4′-trichlorocarbanilide,3,4′,5-tribromosalicylanilide,3-trifluoromethyl-4,4′-dichlorocarbanilide, 8-hydroxyquinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid,1,4-dihydro-1-ethyl-6-fluoro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid, hydrogen peroxide, peracetic acid, phenol, sodium oxychlorosene,parachlorometaxylenol, 2,4,4′-trichloro-2′-hydroxydiphenol, thymol,chlorhexidine, benzalkonium chloride, cetylpyridinium chloride, silversulfadiazine, or silver nitrate.

In some embodiments of the invention, the antiseptic agent is as setforth in the specification of U.S. Provisional Application Ser. No.60/261,447, U.S. Provisional Application Ser. No. 60/316,165, and U.S.Non-Provisional patent application Ser. No. 10/044,842, incorporatedherein by reference in their entirety. Thus, in some embodiments theantiseptic agent comprises a basic reagent and a dye.

The basic reagent may be a guanidium compound, a biguanide, abipyridine, a phenoxide antiseptic, an alkyl oxide, an aryl oxide, athiol, a halide, an aliphatic amine, or an aromatic amine. In somespecific aspects, the basic reagent is a guanidium compound.Non-limiting examples of guanidium compounds include chlorhexidine,alexidine, hexamidine. In other specific embodiments, the basic reagentis a bipyridine. One example of a bipyridine is octenidine. In yet otheraspects, the basic reagent is a phenoxide antiseptic.

The dye may be a triarylmethane dye, a monoazo dye, a diazo dye, anindigoid dye, a xanthene dye, an anthraquinone dye, a quinoline dye, anFD&C dye. Non-limiting examples of triarylmethane dye include gentianviolet, crystal violet, ethyl violet, or brilliant green. Exemplarymonoazo dyes inlude FD&C Yellow No. 5, or FD&C Yellow No. 6. Othernon-limiting examples of FD&C dye include Blue No. 1 or Green No. 3. Onenon-limiting example of diazo dyes is D&C Red No. 17. An example of anindigoid dye is FD&C Blue No. 2. An examples of a xanthene dye is FD&CRed No. 3; of an anthraquinone dye is D&C Green No. 6; and of anquinoline dye is D&C Yellow No. 1.

Other examples of antiseptics that may be used to prepare theantimicrobial solutions of the invention are the phenoxide antisepticssuch as clofoctol, chloroxylenol or triclosan. Still other antisepticagents that may be used to prepare the amntimicrobial solutions of theinvention are gendine, genlenol, genlosan, or genfoctol.

One of skill in the art will appreciate that one can use one or more ofthe antimicrobial agents including one or more antibacterial agent,and/or one or more antifungal agent, and/or one or more antiviral agent,and/or one or more antiseptic agent, and/or combinations thereof.

A wide variety of chelator agents are contemplated as useful inpreparing the antimicrobial solutions of the invention. This includeschelators such as EDTA free acid, EDTA 2Na, EDTA 3Na, EDTA 4Na, EDTA 2K,EDTA 2Li, EDTA 2NH₄, EDTA 3K, Ba(II)-EDTA, Ca(II)-EDTA,Co(II)-EDTACu(II)-EDTA, Dy(III)-EDTA, Eu(III)-EDTA, Fe(III)-EDTA,In(III-EDTA, La(III)-EDTA, CyDTA, DHEG, diethylenetriamine penta aceticacid (DTPA), DTPA-OH, EDDA, EDDP, EDDPO, EDTA-OH, EDTPO, EGTA, HBED,HDTA, HIDA, IDA, Methyl-EDTA, NTA, NTP, NTPO, O-Bistren, TTHA, EGTA,DMSA, deferoxamine, dimercaprol, zinc citrate, a combination of bismuthand citrate, penicillamine, succimer or Etidronate. It is contemplatedthat any chelator which binds barium, calcium, cerium, cobalt, copper,iron, magnesium, manganese, nickel, strontium, or zinc will beacceptable for use in the present invention.

Alternatively, one may use at least one anticoagulant such as heparin,hirudin, EGTA, EDTA, urokinase, streptokinase, hydrogen peroxide etc.,in the preparation of the antimicrobial solutions of the invention.

A variety of alcohols are contemplated as useful in the preparation ofthe instant antimicrobial solution, and include any antimicrobiallyactive alcohol. Non-limiting examples of alcohols include ethanol,methanol, isopropanol, propylene glycol, benzyl alcohol, chlorobutanol,phenylethyl alcohol, and the like. The concentration of the alcohol ispreferably in the range of 5%-80% (v/v), more preferably in the range of10% to 50%, more preferably in the range of 15% to 40%, more preferablyin the range of 20% to 30%, with the most preferable being about 25%.Thus, the more preferred concentration of alcohol will include 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or 80% (v/v) of the alcohol in the preparation of the instantantimicrobial solutions. This includes the use of intermediateconcentrations of alcohol such as 11%, 22.5%, 26% and the like.

One of skill in the art will appreciate that the solutions of theinstant invention can comprise various combinations of at least onealcohol, at least one antimicrobial agent, and at least onechelator/anticoagulant. In some specific embodiments, the solution ofthe invention comprises at least one alcohol, at least one tetracyclineand at least one chelator/anticoagulant. In a specific aspect, such anantimicrobial solution comprises ethanol, at least one tetracycline andEDTA or heparin.

In other specific aspects, such a solution comprises ethanol,minocycline and EDTA or heparin. In one embodiment of this aspect, theconcentration of minocycline is 0.001 mg/ml to 100 mg/ml. In anotherembodiment, the concentration of minocycline is about 3 mg/ml. Inanother aspect, the concentration of EDTA is in the range of 10-100mg/ml. In one embodiment of this aspect, the concentration of EDTA isabout 30 mg/ml.

The invention also provides methods for reducing microbial organismsfrom a surface comprising: a) obtaining an antimicrobial solution of theinvention as set forth above; and b) contacting the surface with theantimicrobial solution, whereby said contacting reduces microbialorganisms from the surface.

In one embodiment of the method, the contacting is performed for 4 hoursor less. In other embodiments of the method, the contacting is performedfor 2 hours or less, for 1 hour or less, for 30 minutes or less, or for15 minutes or less.

In another aspect, the method further comprises eradicating microbesfrom the surface wherein the contacting is performed for about 15minutes or more.

The methods of the invention can be used to reduce microbial agents fromthe surface of a medical device such as a catheter, an endotrachealtube, a nephrostomy tube, a biliary stent, an orthopedic device, aprosthetic valve, a medical implant, dental devices or dental implants,cardiac assist devices, vascular grafts, tracheostomy, ventriclulostomydevices, or intrathecal devices. In some aspects, the catheter is anindwelling catheter such as a central venous catheter, a peripheralintravenous catheter, an arterial catheter, a Swan-Ganz catheter, ahemodialysis catheter, an urinary catheter, a peritoneal catheter, anumbilical catheter, a percutaneous nontunneled silicone catheter, acuffed tunneled central venous catheter or a subcutaneous central venousport.

In other embodiments, the methods of the invention are useful inreducing microbial agents from a surface such as an organic surface oran inorganic surface. An organic surface is exemplified by skin,surgical sutures, mucosal membrane surface, or an epithelial surface. Aninorganic surface may be the surface of a pipe or pipeline, a floor, atable-top, a counter-top, hospital equipment, or a wheel chair, etc.Non-limiting examples of a pipe is an oil pipeline, a water pipeline, anice machine pipe, or a beverage dispensing pipe.

It is contemplated that the antimicrobial solutions of the presentinvention will find particular usefulness as antimicrobial mouthwashsolutions. Such mouthwash solutions are contemplated to be useful bothin conjunction with dental procedures and oral sterilization as well asin general dental and oral hygiene applications. Antimicrobial mouthwashis becoming extremely important in the prevention of oral cavityinfections as well as aspiration pneumonia. Microbial organisms in themouth particularly around the teeth, embed themselves in biofilm and thepathogenesis of infection and colonization is similar to that seen in,for example, vascular catheters. In this regard, it is contemplated thatone will preferably apply the triple combinations of the presentinvention, that will include an antimicrobial (possibly antiseptic) withEDTA and low concentration alcohol as a mouthwash or mouth flushsolution.

The invention also provides a kit for disinfecting a surface to reducemicroorganisms thereon, wherein the kit comprises components includingat least one antimicrobial agent, at least one anticoagulant/chelator,and at least one alcohol, contained in a suitable container. Thecomponents may be combined in a single container, or powdered componentsmay be lyophilized, combined and separately compartmentalized, or all ofthe components may be placed in separate containers. In someembodiments, only the antimicrobial agent(s) is included as a driedpowder. In aspects comprising powdered components, the kit mayoptionally include a second carrier solution for reconstituting thelyophilized antibiotic agent(s).

In preferred aspects, the kit will include a unit dose of apharmacologically effective amount of minocycline and EDTA (or heparin),either provided separately as a lyophilized or powdered dose or alreadymixed in an ethanol solution. In a specific embodiment, the unit dosecontains at least about 9 mg of minocycline and at least about 90 mg ofEDTA. Such a kit may further comprise a preselected amount of an ethanolsolution such that when the ethanol solution is mixed with thelyophilized unit dose, the concentration of minocycline is 3 mg/ml andthe concentration of EDTA is 30 mg/ml.

Kits in accordance with the present invention may be used toreduce/eliminate microbes on the surface of a medical device, a pipe orpipeline, a floor, a table-top, a counter-top, hospital equipment, or awheel chair. It is also contemplated that the kits of the invention willfurther comprise a means for introducing the kit components into themedical device, the pipe or surface.

In some specific aspects of the invention, a syringe or vial comprisinga lyophilized unit dose of a pharmacologically effective amount of oneor more of the three components of the flush solutions of the presentinvention. For example, such a syringe may comprise minocycline and EDTA(or heparin) mixed in an ethanol solution. In a specific embodiment, theunit dose contains at least about 9 mg of minocycline and at least about90 mg of EDTA. Such a syringe or vial may further comprises apreselected amount of an ethanol solution such that when the ethanolsolution is mixed with the lyophilized unit dose, the desiredconcentration of the particular agent is obtained, such as about 3 mg/mlin the case of minocycline and about 30 mg/ml. in the case of EDTA.

In other embodiments of the invention, a locking solution for fillingand/or flushing a medical indwelling device such as, but not limited to,an implanted catheter is contemplated. The locking solution may compriseat least one antimicrobial agent, at least one chelator and/oranticoagulant, and at least one alcohol.

Some of the terms used in the present application are defined below:

An “antimicrobial agent” is defined herein as an agent that hasantibiotic properties against bacteria, fungi, viruses and otherpathogens and includes antibacterial agents, antifungal agents,antiviral agents and antiseptic agents.

As used herein, the term “antifungal agent” is defined as a compoundhaving either a fungicidal or fungistatic effect upon fungi contacted bythe compound. As used herein, the term “fungicidal” is defined to meanhaving a destructive killing action upon fungi. As used herein, the term“fungistatic” is defined to mean having an inhibiting action upon thegrowth of fungi.

As used herein, the term “antibacterial agent” is defined as a compoundhaving either a bactericidal or bacteriostatic effect upon bacteriacontacted by the compound. As used herein, the term “bactericidal” isdefined to mean having a destructive killing action upon bacteria. Asused herein, the term “bacteriostatic” is defined to mean having aninhibiting action upon the growth of bacteria.

As used herein, the term “antiviral agent” is defined as a compound thatcan either kill viral agents or one that stops the replication ofviruses upon contact by the compound.

For the purposes of this disclosure, the phrase “effective amount” or“therapeutically effective amount” is defined as a dosage sufficient toinduce a microbicidal or microbistatic effect upon the microbescontacted by the composition on a surface.

The phrase “a chelator” denotes one or more chelators. As used herein,the term “chelator” is defined as a molecule comprising nonmetal atoms,two or more of which atoms are capable of linking or binding with ametal ion to form a heterocyclic ring including the metal ion.

As used herein the terms “contact”, “contacted”, and “contacting”, or“exposed” and “exposure” are used to describe the process by which anyof the compositions disclosed in the present invention, comes in directjuxtaposition with the surface of a medical device or any other surfacefrom which microbial growth is to be reduced or eradicated.

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Ethanol in combination with M-EDTA as a flush solution used for15 minutes or 24 hours, as indicated, tested against MRSA in biofilm.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Microorganisms that attach themselves to inert surfaces, such as medicaldevices including, vascular catheters, endotracheal tubes, Foleycatheters, biliary stents, nephrostomy tubes, prosthetic valves,ventriculostomy or epidural catheters, or fluid pipelines, such as oilpipelines or water pipelines, produce a layer made of exopolysaccharidecalled microbial biofilm. These organisms embed themselves in thislayer. This biofilm layer ultimately becomes the protective environmentthat shields these organisms on the inert surface from the antimicrobialactivity of various antibiotics or antiseptics. In U.S. Pat. Nos.5,362,754 and 5,688,516, incorporated herein by reference in theirentirety, the present inventor demonstrated that a combination of one ormore antimicrobial agent with one or more chelator and/or anticoagulant(such as EDTA or heparin) reduces or eradicates theseantibiotic-resistant biofilm embedded microorganisms if theantimicrobial and chelator combination is allowed to dwell on thesurface for at least 4 hours. However, in both clinical andenvironmental situations, it is typically not feasible to allow a 4 hourdwell time for the chelator and antimicrobial agent to reduce oreradicate the microbes. For example, it is not possible to interrupt thetherapy of critically ill patients receiving continuous infusion therapythrough a vascular catheter for 4 hours. It is also not possible tointerrupt an environmental situation involving fluid pipelines for 4hours to allow for such a prolonged dwell time of antimicrobial/chelatorsolution.

A. The Present Invention

The present invention allows rapid reduction and/or eradication ofmicroorganisms embedded in a biofilm in a time as short as about 15minutes of exposure to combinations of at least one antimicrobial and atleast one chelator/anticoagulant, if this combination is prepared in analcohol. This is exemplified in one embodiment by minocycline-EDTA in anethanol solution, which is described in detail in the application.However, one of skill in the art will recognize that one may use anyantimicrobial agent, any chelator/anticoagulant and any alcohol.

In addition, the present invention provides antimicrobial solutionscomprising one or more antimicrobial agents, one or morechelator/anticoagulant, and an alcohol solution. The present inventionalso provides methods for the rapid reduction or eradication ofmicroorganisms embedded in a biofilm on a surface comprising contactingor exposing the surface to a flush solution of the invention. Thus, theinvention provides methods for reducing or eradicating microbes from thesurfaces of medical devices, including indwelling medial devices, aswell as other surfaces, pipelines and the like.

The compositions and the methods of the present invention have anunexpected and surprising efficacy not provided by compositions thatcomprise only alcohol solutions, or compositions that comprisecombinations of antimicrobials with chelators/anticoagulants. In onespecific example, the combination of the antimicrobial agent minocyclinewith the chelator/anticoagulant EDTA requires about 4 hours of exposureor dwell time to reduce microbes from the surface of a medical device.On the other hand, a 25% ethanol solution alone suppresses colonizingorganisms embedded in biofilm, but does not eradicate them. However, oneexemplary composition of the present invention, comprising minocycline,EDTA and 25% ethanol provides rapid reduction and/or eradication of themicrobial organisms within 15 minutes of exposure and also prevents there-growth of the microbes.

1. Medical Applications

One of the applications of the antimicrobial flush solutions of theinvention is to reduce or eradicate microbes from the surfaces ofmedical devices especially indwelling medical devices such as catheters,endotracheal tubes, nephrostomy tubea, biliary stents, orthopedicdevices, prosthetic devices, and medical implants.

There are at least 5 million central venous catheters inserted annuallyin the United States, 1.5 million of which are long-term catheters thatremain in place for an average of 100 days, and at least 3.5 millionshort-term catheters that remain for an average of 7 days. All of thesevenous catheters are flushed with heparin on a daily basis. It isestimated that at least 150-175 million catheter flushes occur annuallyin the United States alone. Heparin has good anticoagulant activity and,hence, prevents thrombotic occlusions. However, heparin has noantimicrobial activity and, in fact, given the alkaline media thatheparin creates, it has been shown to be a promoter of microbialcolonization of catheter surfaces. Irrespective of whether heparin isused, almost 90%-100% of indwelling vascular catheters end up beingcolonized with organisms embedded in biofilm on the surface of thesedevices, particularly at the lumenal surface. Hence, the most seriousand frequent complication of vascular catheters is infection, whereby asfluid is flushed through the lumen of the catheter, microorganismsmigrate into the bloodstream and cause catheter-related bloodstreaminfections. Indwelling central venous catheters are associated witharound 5%-8% frequency of catheter-related bloodstream infection, whichin turn is associated with an attributable mortality of 25% incritically ill patients. Such an event is also associated with highmorbidity and a cost per episode of an average of $30,000.

EDTA is a well-known chelator of iron and calcium, as well as an activeanticoagulant used in blood collection tubes. EDTA has been shown tohave equivalent anticoagulant activity to heparin. In addition, EDTA hasantibiofilm activity and enhances the antimicrobial activity of otherantimicrobial agents, such as minocycline. However, for a combination ofan antmicrobial with a chelator (such as minocycline-EDTA) to eradicateorganisms embedded in biofilm, contacting the surface for at least4-hour with this combination is required. This is demonstrated in U.S.Pat. No. 5,362,754 (see especially data in Tables 3, 4 and 5) and inU.S. Pat. No. 5,688,516, (see especially columns 15 and 16 and Tables 3,4 and 5). This prolonged period of contacting or dwell time is notpossible in the highest risk patient population (i.e., in patientsreceiving total parental nutrition or critically ill patients), as thesepatients require a continuous, often uninterrupted, infusion through thecatheter. In order to allow for a rapid reduction or eradication ofmicroorganisms, an improvement has been developed in the presentinvention wherein the antimicrobial(s) and chelator(s)/anticoagulant(s)is prepared in an alcohol solution. In one example, this is embodied byaminocycline-EDTA combination in a 25% ethanol solution.

The present invention, thus, provides that indwelling medical devicessuch as catheters be flushed with this antimicrobial andchelator/anticoagulant in an alcohol-based solution. This will providechelation/anticoagulation through the chelator (such as EDTA). Inaddition, the combination of antibiotic/chelator with an alcohol resultsin broad-spectrum reduction or eradication of microbial organismsembedded in biofilm. The alcohol further increases the efficacy of thecombination.

Some examples of indwelling medical devices that may be treated with thesolutions of the present invention include abdominal cavity drainagebags, connectors and tubing used by colostomy patients, vascular shunts,orthopedic, intraocular, or penile prosthesis devices. Angioplastydevices, heart valves and cardiac pacemakers also are included withinthe present invention. Catheters such as urinary, venous, arterial, andperitoneal catheters may be treated with the flush solutions of theinvention. In addition, tracheotomy devices, shunts, surgical sutures,and other medical devices or prosthesis can be treated.

Furthermore, the medical devices which are amenable to coatings of thecompositions of the invention generally have surfaces composed ofthermoplastic or polymeric materials such as polyethylene, Dacron,nylon, polyesters, polytetrafluoroethylene, polyurethane, latex,silicone elastomers and the like. Devices with metallic surfaces arealso amenable to coatings with the antibiotic combinations. Such devicesare exemplified by bone and joint prosthesis. It is also contemplatedthat the solutions of the invention will be used to disinfect organicsurfaces such as skin as well as mucosal surfaces.

An antimicrobial locking solution of the present invention may compriseat least one alcohol, at least one antimicrobial agent and at least onechelator and/or anticoagulant. Various antimicrobial substances asdisclosed herein and that are well known to one of ordinary skill in theart may be combined with the locking solution in order to inhibitinfection. The antimicrobial locking solution of the present inventionmay be use for filling or flushing a medical device such as anindwelling device such as an implanted catheter. Other medical devicesthat are contemplated for use in the present invention are disclosedherein.

2. Environmental Applications

Other than reduction/eradication of microbes in medical devices, theflush solutions of the present invention are also useful in theeradication of the surfaces of other surfaces that microbes can grow onsuch as pipes, pipelines etc. Fluid pipelines, such as oil and waterpipelines, are often obstructed by lumenal biofilm that is produced bymicroorganisms that colonize the internal surface of these pipelines.Often these pipelines are flushed with antimicrobial agents. However,antimicrobial and antiseptic agents have little activity againstorganisms embedded in biofilm. Tons of antibiotics, such as gentamicin,are often used to flush the lumen of oil pipelines, to no avail. Thepresent invention provides new and effective compositions and methodsfor the eradication of organisms, as well as biofilm embedding the lumenof pipelines (oil, water), as well as other devices, such as icemachines. These pipelines or machines can be flushed or rinsed with thecompositions of the invention that comprise at least one antimicrobialagent and at least one chelator or anticoagulant prepared in a basesolution of ethanol. Flushing the pipelines, machines or tubes with thecompositions of the invention provide rapid reduction and/or eradicationof the biofilm and the organisms in biofilm thereby preventing anyobstruction or contamination of the water, oil or the ice machines incertain environmental settings.

B. Antimicrobial Agents and Microbes

The present compositions are contemplated to have one or moreantimicrobial agents. “Antimicrobial agents” are defined herein asantibacterial agents, antifungal agents, antiviral agents and/orantiseptic agents.

While the invention is not limited to any particular antimicrobial agentsome exemplary classes and examples of antibacterial agents, antifungalagents, antiviral agents as well as antiseptic agents are describedabove in the section entitled “summary of invention.” Of course one ofskill in the art will appreciate that any combination as well as agentsfrom the different types and classes of the antimicrobial agents can becombined to prepare the solutions of the invention.

Some non-limiting exemplary bacterial and fungal microbes that can bereduced or eradicated by the compositions and methods of the inventioninclude Staphyloccous species such as Staphylococcus epidermidis,Staphylococcus aureus; Aspergllus species, such as Aspergillus flavus,Aspergillus terreus; Fusarium oxysporum, Candida species, such asCandida krusei, Candida parapsilosis, Candida tropicalis, Candidaalbicans and Candida glabrata. In addition, viruses can also beeradicated.

C. Chelators and/or Anticoagulants

A chelate is the type of coordination compound in which a central metalion is attached by coordinate links to two or more nonmetal atoms in thesame molecule. Heterocyclic rings are thus formed during chelation, withthe metal atom as part of the ring. The molecule comprising the nonmetallinking atoms is termed a chelator. Chelators are used in variouschemical applications, for example as titrating agents or as metal ionscavengers. Chelators can be used to remove ions from participation inbiological reactions. For example, the well-known chelatorethylenediamine-N,N,N′,N′,-tetraacetic acid (EDTA) acts as ananticoagulant because it is capable of scavenging calcium ions from theblood.

It has been previously shown that chelators have significant growthinhibitory effect against several microbes. It is known that iron andother trace metals are essential in the life cycle of microorganismssuch as fungi and bacteria. Without these trace metals, microbes areunable to grow and reproduce. Although iron is abundant in nature, itsavailability for microbial assimilation is limited owing to theinsolubility of ferric ions at neutral or alkaline pH. As a consequence,many microbes have evolved their own specialized trace metal-scavengingmolecules, called siderophores, which bind with trace metals and makethem available for uptake by the microbes. The chelators used inconjunction with the present invention provide an inhibitory effect uponmicrobial pathogens by competing with the siderophores for any availabletrace metal ions. In this way, the chelators present in thepharmaceutical preparations of the present invention “steal” the metalions essential for microbial growth, effectively causing the microbe to“starve to death.” The additional antibiotic agents and the ethanol ofthe compositions of the present invention then come in and attack theweakened microbe, thereby destroying them or inhibiting their growth.

Table 1 below provides a representative list of chelators useful inconjunction with the present invention. However, the list provided inTable 1 is not meant to be exhaustive. Preferred chelators are thosewhich bind trace metal ions with a binding constant ranging from 10¹ to10¹⁰⁰. More preferred chelators are those which bind trace metal ionswith a binding constant ranging from 10¹⁰ to 10⁸⁰; and most preferredchelators are those which bind trace metal ions with a binding constantranging from 10¹⁵ to 10⁶⁰. Furthermore, preferred chelators are thosewhich have been shown to have an inhibitory effect upon target microbialpathogens, for example the disodium salt of EDTA.

TABLE 1 Chelators Abbreviation Full Name EDTA freeEthylenediamine-N,N,N′,N′,-tetraacetic acid acid EDTA 2NaEthylenediamine-N,N,N′,N′,-tetraacetic acid, disodium salt, dihydrateEDTA 3Na Ethylenediamine-N,N,N′,N′,-tetraacetic acid, trisodium salt,trihydrate EDTA 4Na Ethylenediamine-N,N,N′,N′-tetraacetic acid,tetrasodium salt, tetrahydrate EDTA 2KEthylenefisminr-N,N,N′,N′-tetraacetic acid, dipotassium salt, dihydrateEDTA 2Li Ethylenediamine-N,N,N′,N′-tetraacetic acid, dilithium salt,monhydrate EDTA 2NH₄ Ethylenediamine-N,N,N′,N′-tetraacetic acid,diammonium salt EDTA 3K Ethylenediamine-N,N,N′,N′-tetraacetic acid,tripotassium salt, dihydrate Ba(II)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, barium chelate Ca(II)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, calcium chelate Ce(III)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, cerium chelate Co(II)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, cobalt chelate Cu(II)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, copper chelate Dy(III)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, dysprosium chelateEu(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, europiumchelate Fe(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, ironchelate In(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, indiumchelate La(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid,lanthanum chelate Mg(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraaceticacid, magnesium chelate Mn(II)-EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, manganese chelateNi(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, nickel chelateSm(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, samariumchelate Sr(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid,strontium chelate Zn(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraaceticacid, zinc chelate CyDTA trans-1,2-Diaminocyclohexane-N,N,N′,N′-tetraaceticacid, monohydrate DHEG N,N-Bis(2-hydroxyethyl)glycine DTPA-OH1,3-Diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid DTPA1,3-Diaminopropane-N,N,N′,N′-tetraacetic acid EDDAEthylenediamine-N,N′-diacetic acid EDDP Ethylenediamine-N,N′-dipropionicacid dihydrochloride EDDPO Ethylenediamine-N,N′-bis(methylenephosphonicacid), hemihydrate EDTA-OHN-(2-Hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid EDTPOEthylenediamine-N,N,N′,N′-tetrakis(methylenephosponic acid) EGTAO,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′- tetraacetic acid HBEDN,N-diacetic acid HDTA 1,6-Hexamethylenediamine-N,N,N′,N′-tetraaceticacid HIDA N-(2-Hydroxyethyl)iminodiacetic acid IDA Iminodiacetic acidMethyl-EDTA 1,2-Diaminopropane-N,N,N′,N′-tetraacetic acid NTANitrilotriacetic acid NTP Nitrilotripropionic acid NTPONitrilotris(methylenephosphoric acid), trisodium salt O-Bistren7,19,30-Trioxa-1,4,10,13,16,22,27,33-octaabicyclo [11,11,11]pentatriacontane hexahydrobromide TTHATriethylenetetramine-N,N,N′,N″,N′″,N′″-hexaacetic acid

In addition, as several anticoagulants have similar chelating and henceantimicrobial activity use of anticoagulants such as EGTA, EDTA,heparin, urokinase, streptokinase, low molecular weight heparin,enoxaparin, sodium coumarin, indanedione, anisindione, warfarin,protamine sulfate, anti-thrombin III, nitrilotriacetic acid, potassiumsodium tartrate, potassium hydrogen D-tartrate, L-tartaric aciddipotassium salt, L-tartaric acid disodium salt, L-tartaric acidmonosodium salt, tris(carboxymethyl)amine, warfarin, acetylsalicylicacid, ibuprofen, indomethacin, prostaglandins, sulfinpyrazone,streptokinase, urokinase, tissue plasminogen activator, coumarin,protamine sulfate, anti-thrombin III, coumadin, protein C/protein S,nicoumalone, phenprocoumon, hirudin, hirulog, or glycosaminoglycans etc.is also contemplated in the present invention. Moreover, additionalchelators, anticoagulants and/or additional agents useful in thepractice of the present invention may be found in U.S. Pat. No.5,688,516, incorporated herein by reference.

D. Alcohols

The flush solutions of the instant invention are contemplated tocomprise an alcohol, such as an antiseptic or disinfectant alcohol.Exemplary alcohols include ethanol, methanol, isopropanol, benzylalcohol, chlorobutanol, phenylethyl alcohol,2-bromo-2-nitropropan-1,3-diol, and the like. The present inventioncontemplates any effective concentration of alcohol, but will typicallyemploy a final alcohol concentration in the range of 5%-80% (v/v), morepreferably in the range of 10% to 50%, more preferably in the range of15% to 40%, more preferably in the range of 20% to 30%, with the mostpreferable being about 25%. Thus, the more preferred concentration ofalcohol will include 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (v/v) of the alcohol inthe preparation of the instant antimicrobial solutions. This includesthe use of intermediate concentrations of alcohol such as 11%, 22.5%,26% and the like.

Alcohols such as ethanol are long known to have disinfectant properties.In EP1245247 and U.S. Pat. No. 6,350,251, it is reported that thecombination of ethanol with EDTA provides a biocidal lock for indwellingmedical devices. In contrast, it has also been shown that a combinationof ethanol with EDTA is less effective in killing microbes than ethanolalone (Sherertz et al., 2002). Thus, the art is in a flux about theexact role of the combination of ethanol with EDTA.

The present inventor has shown that ethanol alone, while requiring onlya relatively short duration of contact, is only partially effective inkilling or controlling microbes on the surface of an indwelling medicaldevice or other surface. In contrast, a combination of an antimicrobialagent and a chelator such as EDTA may be effective, yet it requires asomewhat longer duration of contact (e.g., sometimes on the order of 4hours). However, in the present invention it is shown that the triplecombination of an alcohol, an antimicrobial and a chelator/anticoagulantprovides unexpectedly effective anti-microbial properties in a veryshort duration and in addition to eradicating microbes rapidly from asurface they also preventing re-growth of the microbial pathogen on thesurface. An additional advantage for the triple combination, as shown inthe studies set forth herein below, is that it is effective ateradicating a broader range of microbial organisms (bacteria and fungi),even at the shorter durations of contact with the treated surface.

E. Additional Agents

It is also contemplated that any additional pharmacologically activeingredients or sterilization agents may be comprised in the solutions ofthe present invention or may be used separately for flushing or treatingthe devices of the present invention to further reduce or eliminatepathogenic microbes and viruses. Typical pharmacologically activeingredients include antifibrin agents, anti-thrombotic agents, andanti-inflammatory agents. Anti-inflammatory agents include steroids, andnonsteroidal anti-inflammatory agents, and salicylates. Anti-thromboticdrugs including acetylsalicylic acid, dipyridamole, heparin, ibuprofen,indomethacin, prostaglandins, sulfinpyrazone, warfarin, thrombolyticenzymes such as streptokinase, urokinase, or plasminogen activator.Complexing agents such as ammonium-1-pyrrolidine dithiocarbanate mayalso be used. However, the above examples are not meant to be limiting.

In certain applications, it will be sufficient to provide a singlepharmacologically active ingredient in the device. In other situations,it will be desirable to combine compatible ingredients. For example, itmay prove useful to provide an antimicrobial agent along with ananticoagulant and/or an anti-inflammatory agent. In another example, itmay prove useful to provide multiple antimicrobial agents with differingtarget specificities, modes of action or duration, either alone or incombination with anticoagulants or antiinflammatory agents.

F. Packaging and Kits

Described herein are various packaging techniques that may be employedin providing the flush solutions of the invention as part of acommercially available kit. The kit will optionally include aninstruction sheet insert to identify how the kit is to be used.

The kits described in this section are exemplified by a solutioncomprising minocycline as the antibiotic, EDTA as thechelator/anticoagulant, and ethanol. However, as will be appreciated bythe skilled artisan, any other combination of one or more antibiotic,one or more chelator/anticoagulant, and ethanol as described in thepresent disclosure may be packaged in a similar manner. The kit maycomprise of one or two or three or more compartments. The components ofthe kit may be provided in separate compartments or in the samecompartment. The components of the kit may be provided separately ormixed. The mixed components may contain two or more agents such as anantibiotic, a chelator/anticoagulant, or ethanol, or additionalcomponent.

One of the packaging options below maintain the ingredients, forexample, the antibiotic, such as minocycline, and the chelatingagent/anticoagulant, such as EDTA, in an uncombined form. Thesecomponents are to be combined shortly before use. These packagingoptions are contemplated to be part of a 2-compartment orthree-compartment container system to provide a total volume of about 3ml of the ready to use preparation. Any compartmentalized containersystem may be used to package the compositions of the present invention.An exemplary container system is available from Becton Dickinson.

Option 1: A 3-Compartment system comprising two dry components such as3-9 mg minocycline (dry), 10-100 mg EDTA (powdered) and one wetcomponent comprising 3 ml diluent (alcohol alone or diluted in saline ordistilled water). When ready for use, the dry components, minocyclineand EDTA, will be allowed to mix with the diluent. Final concentrationof the mixture should be about 3 mg/ml minocycline and 30 mg/ml EDTA.

Option 2: A 2-Compartment system antibiotic and chelator/anticoagulant(one wet, one dry) comprising for example 3-9 mg/ml minocycline and10-100 mg EDTA. When ready for use, the dry EDTA powder will be combinedwith the minocycline in solution. The minocycline may be suspended ineither saline, distilled water, alcohol solution or otherphysiologically acceptable diluent. Alternatively, the minocycline maybe in a dry powdered form, and the EDTA in solution. A wet/wet® dualchamber container system, available from Becton-Dickinson, may be usedin these applications.

Option 3: A 2 compartment system comprising both wet compartmentscomprising antimicrobial agent(s) and chelator/anticoagulant comprisingin one example 10-100 EDTA Solution and 3-9 mg/ml Minocycline Solutionwhere the solution comprises alcohol. When ready for use, the EDTAsolution will be combined with minocycline solution. Once combined, thesolution will have a concentration of 3 mg/ml minocycline and 30 mg/mlEDTA. A wet/wet® dual chamber container system, available fromBecton-Dickinson, may be used in these applications.

Option 4: A 2 compartment system both comprising dry powders of theantimicrobial agent(s) and chelator/anticoagulant in a diluentcomprising for example, 10-100 EDTA (dry) and 3-9 mg minocycline (dry)and diluent solution. The dry EDTA and dry minocycline may be suspendedin a solution of an alcohol made in either saline, distilled water, orother physiologically acceptable diluent. A liquid/dry® dual containersystem, from Becton-Dickinson, may be used. When ready for use, the dryminocycline powder will be allowed to combine with the EDTA solution.The EDTA can be suspended in either saline or distilled water, oralcohol solution, or other physiologically acceptable diluent.

The various compartmentalized embodiments of the present invention asdisclosed above, may be provided in a kit form. Such kits would includea container means comprising a volume of diluent, comprising an alcoholoptionally diluted if required in a solution such as saline or sterilewater, a second (or more) container means comprising one or moreantimicrobial or biocide, a third (or more) container means comprisingone or more chelating/anticoagulant agent. The dry components mayoptionally be mixed in one compartment. The addition of the diluentwould then be performed immediately prior to use.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antimicrobial/chelator/anticoagulant/alcohol may be placed,and preferably, suitably aliquoted. Where a second or third antibioticagent, other chelator, alcohol, or additional component is provided, thekit will also generally contain a second, third or other additionalcontainer into which this component may be placed. The kits of thepresent invention will also typically include a means for containing thealcohol, antimicrobial agent, chelator/anticoagulant, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic, or glasscontainers into which the desired vials are retained.

G. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Reduction and/or Eradication of Microbes Using the ModifiedRobbins Device Model

In vitro Model of Colonization (Modified Robbins Device). The in vitromodel utilized a modified Robbins device (MRD) to study the colonizationof catheter segments with organisms embedded in biofilm. The modifiedRobbins device has been previously described (Nickel et al., 1991; Evanset al., 1987, see also U.S. Pat. No. 5,362,754) and is constructed froman acrylic block, 42 cm long with a lumen of 2×10 mm. It consists of 25evenly spaced specimen plugs, each connected to a silicone cathetersegment (Allegiance Healthcare Corp., McGaw Park, Ill.) whose anteriorsurface (0.3 cm²) comes in contact with the flushed infusate. Afterplacing the catheter segments in the specimen plug of the modifiedRobbins device, the entire apparatus was gas sterilized using ethyleneoxide. A 500 ml 5% dextrose in water (5% D₅/W) was connected to themodified Robbins device through an intravenous tubing administration setand was subsequently infected with an innoculum of 10⁸ CFU/ml ofmethicillin-resistant Staphylococcus aureus (MRSA), to produce aninfected infusate at the concentration of 2×10⁵ CFU/ml. Thebiofilm-producing S. aureus isolates were obtained from patients withCRBSI. In another series of experiments, 500 ml 5% D₅/W bag was infectedwith a biofilm-producing C. parapsilosis using an inoculum of 10⁵ CFU/mlto produce an infected infusate at a concentration of 2×10² CFU/ml of C.parapsilosis. The whole system was incubated at 37° C. and the infectedinfusate was flushed through the MRD using a peristaltic pump permittingthe infusate to flow at the rate of 60 ml/hour for 8 hours. The modifiedRobbins device was left to incubate for a total of 18 hours (another 10hours). Subsequently, the infected bag was removed and a 250 ml salinesterile bag which was infused through the MRD at 125 ml/hour for 2 hoursin order to remove all free floating organisms. To insure biofilmformation, at least three catheter segments were randomly removed fromthe 25 evenly spaced catheter segments in the MRD and studied byscanning electron microscopy. This was repeated for every organismtested.

Exposure to Anticoagulants/Antimicrobials. The remaining cathetersegments were removed and each segment was placed in a tube containing 2ml of one of the following broth solutions: (1) Mueller-Hinton broth(Becton Dickinson & Co., Cockeysville, Md.); (2) EDTA at a concentrationof 30 mg/ml in broth (Abbott Laboratories, North Chicago, Ill.); (3)minocycline at 3 mg/ml in broth (Wyeth-Ayerst Laboratories,Collegeville, Pa.); (4) minocycline (3 mg/ml) and EDTA combination(M-EDTA) in broth; (5) 25% ethanol (ETOH) solution in broth; (6)minocycline at 3 mg/ml in 25% ethanol solution in broth; (7) EDTA 30mg/ml in 25% ethanol solution in broth; and (8) M-EDTA in 25% ethanolsolution in broth. The experiments were repeated in triplicate orquadruplicate, and during each experiment, 2-5 catheter segments wereexposed to the same solution for only 15 minutes at 37° C. Subsequently,some of the catheter segments were immediately removed and cultured byscrape sonication. Other alternating catheter segments were removed,placed in broth (TSB), incubated for 24 hours, and then cultured byscrape sonication. This added step of re-incubating catheter segments inbroth after the 15 minutes exposure was done to determine whether theseagents suppressed the growth of organisms embedded in biofilm oreradicated them. The surface of the catheter segment that was exposed tothe infected infusate was scraped with a sterile wooden applicator stickand placed, along with the stick, in a tube containing 0.5 ml oftrypticase soy broth. The tubes were sonicated for five minutes; 0.1 mlof the sonicated broth solution in the tube was pipetted and plated overa blood agar plate, which was incubated at 37° for 24 hours. The agarplates were checked for any contaminants. The isolated organisms had tobe of the same species and colonial morphology as the original organismused to infect the infusate. The number of colonies quantitated from theagar plate was multiplied by five to correct for the dilution factor andto determine the total number of colonies isolated from a particularcatheter segment. A confluent growth of 100 or greater was calculated as≧500 colonies.

Definitions. Inhibitory activity or suppression is defined as no growthof microbial organisms immediately after 15 minute exposure to theantimicrobial solution. However, regrowth of the organisms after 24hours incubation in broth was observed.

Eradication is defined as no growth of organisms after immediate 15minutes exposure to the antimicrobial solution with no subsequent growthupon reincubation for 24 hours in broth.

Results

As shown in Table 2, EDTA alone failed to eradicatemethicillin-resistant S. aureus and C. parapsilosis organisms embeddedin biofilm after 15 minutes of exposure, resulting in re-growth after 24hours of incubation of the catheter segments in broth solution.Minocycline alone (at a concentration of 3 mg/ml) with or without EDTAresulted in some decrease in colonization. However, organisms continuedto grow after 15 minutes of exposure and after 24 hours re-incubation inbroth at 37° C. A 25% ethanol solution suppressed growth initially to amean concentration level of 138 colony forming units (CFU). However,upon re-incubation in broth at 37° C. for 24 hours, there was completere-multiplication and growth of the staphylococcal organisms embedded inbiofilm to a high level of 500 CFU per catheter segment, which iscomparable to the growth of control catheter segments. The combinationof EDTA and 25% ethanol solution resulted in a significant decrease incolonization immediately after 15 minutes of exposure to this solution.However, regrowth occurred after re-incubation in broth solution at 37°C. for an additional 24 hours. Minocycline in 25% ethanol, with orwithout EDTA, resulted in complete eradication of microorganismsembedded in biofilm after 15 minutes of exposure to the solutions. Inaddition, re-incubation of the catheter segments in broth for anadditional 24 hours at 37° C. failed to allow regrowth of the organisms,verifying the complete eradication of the S. aureus organisms embeddedin biofilm.

TABLE 2 Modified Robbins Device MRSA Model 24 h growth 15 minutes after15 min Drug Combination in Drug mean in drug mean Tested CFU MRSA CFUMRSA 3 mg/ml minocycline 323.7 ± 199.0 308.3 ± 151.1 30 mg/ml EDTA 479.6± 44.9  500.0 ± 0.0  25% EtOH in MHB 138.0 ± 193.8 500.0 ± 0.0  3 mg/mlminocycline/30 mg/ml 295.0 ± 182.0 170.8 ± 149.1 EDTA 25% EtOH/30 mg/ml25.8 ± 63.5 333.9 ± 234.9 EDTA 25% EtOH/3 mg/ml 0 0 minocycline 25%EtOH/30 mg/ml 0 0 EDTA/3 mg/ml minocycline Control (MH Broth alone)440.3 ± 113.4 500.0 ± 0.0 

As shown in Table 3, EDTA alone, minocycline alone and M-EDTA failed toeradicate C. parapsilosis organisms embedded in biofilm. A 25% ethanolsolution with or without minocycline, inhibited C. parapsilosis growthafter 15 minutes of exposure. However, regrowth was noted after 24 hoursincubation in broth. EDTA in 25% ethanol and M-EDTA in 25% ethanolcompletely eradicated C. parapsilosis in biofilm after 15 minutesexposure with no regrowth after reincubation in broth.

TABLE 3 Modified Robbins Device Candida Parapsilosis Model 24 h Growthafter 15 15 Minutes in Drug Minutes in Drug Drug Combinations Mean CFUMean CFU Tested C. Parapsilosis C. Parapsilosis 3 mg/ml minocycline138.3 ± 111.8 500.0 ± 0.0  30 mg/ml EDTA  160 ± 78.8 500.0 ± 0.0  25%EtOH in MHB 0 142.9 ± 225.9 3 mg/ml minocycline/30 mg/ml 152.5 ± 161.3500.0 ± 0.0  EDTA 25% EtOH/30 mg/ml 0 0 EDTA 25% EtOH/3 mg/ml 0  83.3 ±186.3 minocycline 25% EtOH/30 mg/ml 0 0 EDTA/3 mg/ml minocycline Control(MH Broth alone) 500.0 ± 0.0  500.0 ± 0.0 

Minocycline alone, EDTA alone or the combination of minocycline and EDTAfailed to eradicate organisms embedded in biofilm after a rapid exposureof only 15 minutes. 25% ethanol solution also failed to eradicateorganisms embedded in biofilm and a high level of regrowth was apparentafter catheter segments were re-incubated in broth for an additional 24hours at 37° C.

The combination of ethanol/EDTA did achieve inhibition or suppression oforganisms embedded in biofilm after 15 minutes of exposure of thecatheter surfaces to this solution. However, regrowth was noted uponre-incubation of the catheter segments in broth for 24 hours at 37° C.The combination of EDTA/25% ethanol, however, was superior in itsinhibitory activity when compared to 25% ethanol alone.

The combination of minocycline in 25% ethanol with or without EDTA washighly active in eradicating organisms embedded in biofilm after 15minutes of exposure to this combination. Regrowth of C. parapisilosisoccurred occasionally after exposure to minocycline in 25% ethanol.Regrowth failed to occur after exposure to M-EDTA in 25% ethanol,verifying the complete eradication of S. aureus organisms embedded inbiofilm after rapid exposure to this triple combination.

Because EDTA has anticoagulant activity and, in these experiments, seemsto have added to the antimicrobial activity of 25% ethanol, it wasprudent to use the triple combination of minocycline/EDTA in 25% ethanolas a flush or antibiotic lock solution of central venous catheters. Incontrast, vancomycin alone or in combination with heparin failed toeradicate microbial organisms embedded in biofilm from cathetersurfaces, even after a dwell time of 4-24 hours (see U.S. Pat. No.5,362,574, columns 11 and 12, Tables 3, 4 and 5.)

Example 2 Testing M-EDTA in 25% Ethanol Using the Silicone DiskBioprosthetic Colonization In Vitro Model

The inventor next determined the efficacy of minocycline and EDTAcombination in 25% ethanol in eradicating staphylococci and candidaembedded in biofilm. Prevention of regrowth after reincubation wasassessed using a novel silicone disk bioprosthetic colonization model.The procedure is described below.

Experimental Procedure

On day 1, pieces of biofilm were prepared. Sterile (Ethylene Oxide GasSterilized) silicone disks were placed in 5 ml sterile snap top Falcontubes and 0.5 ml of pooled plasma added. This was followed by incubation(while rocking) overnight at 37° C.

On day 2, the bacteria was added to form the biofilm. Using sterileplastic transfer pipettes, the plasma was suctioned out from the tubesand replaced with 0.5 ml of bacterial inoculum (50 ml of Mueller-Hintonbroth containing 4-5 colonies of freshly grown bacteria). The tubes wereincubated overnight at 37° C.

On day 3, a drug was added in an attempt to kill the bacteria. Beforeadding the drug, the pieces were washed in 0.5 ml of 0.9% saline inorder to remove any planktonic bacteria. The tubes (containing thebiofilm disks and saline) were placed in the incubator at 37° C. for 30minutes. The saline was then pipetted out using sterile plastic transferpipettes (taking care not to disturb the pieces too much). The siliconedisks were then transferred to new 5 ml snap-top falcon tubes containing0.5 ml of the drug solution to be tested. The drug solutions tested wereas follows: (1) minocycline 3 mg/ml; (2) EDTA 30 mg/ml; (3) 25% ethanolsolution; (4) EDTA 30 mg/ml in 25% ethanol; (5) minocycline 3 mg/ml in25% ethanol; (6) minocycline 3 mg/ml with EDTA 30 mg/ml; and (7) triplecombination of minocycline 3 mg/ml and EDTA 30 mg/ml in 25% ethanolsolution. The disks were allowed to sit in the drug for 1 hour. The drugwas then suctioned out using a plastic transfer pipette. The pieces wereonce again washed with 0.5 ml saline (added, and shaken for 30 seconds).The disks were then transferred to 15 ml snap-top falcon tubescontaining 5 ml of 0.9% saline. The pieces were sonicated for 5 minutes,and then vortexed for 30 seconds. 100 microliters (μl) of the saline wasthen plated on a room temperature TSAII blood agar plate, and evenlyspread using a sterile glass spreader. The plates were incubatedovernight at 37° C.

For the 24 hour reincubation studies, pieces of biofilm were preparedthe same exact way as the regular pieces. Before adding the drug, thepieces were washed in 0.5 ml of 0.9% saline in order to remove anyplanktonic bacteria. The tubes (now containing the biofilm disks andsaline) were placed in the incubator at 37° C. for 30 minutes. Thesaline was then pipetted out using sterile plastic transfer pipettes(taking care not to disturb the pieces too much). The silicone diskswere then transferred to new 5 ml snap-top falcon tubes containing 0.5ml of the drug to be tested. The disks were allowed to sit in the drugfor 15 minutes. The drug was then suctioned out using a plastic transferpipette. The pieces were once again washed with 0.5 ml saline (added,and shaken for 30 seconds). The pieces were then transferred to newsterile 5 ml snap-top falcon tubes containing 0.5 ml of steriletrypticase soy broth (TSB) and then placed in the incubator at 37° C.overnight.

On day 4, colonies were counted and the results recorded. The colonieswere hand counted, and counting was stopped at 100 colonies. Anythinggreater was considered >100 colonies. The counts were recorded, andmultiplied by a factor of 50 because of the dilution factor between the5 ml of saline containing the disk and the 100 μl that was plated ontothe TSAII blood agar plates. The pieces were then sonicated (in the sameTSB that grew overnight) for 5 minutes. 100 μl was then plated on TSAIIblood agar plates, and the plates were placed in the incubator to growovernight at 37° C.

On day 5, the regrowth pieces were counted. The colonies were handcounted, and counting was stopped at 100 colonies. Anything greater wasconsidered >100 colonies. The counts were recorded, and multiplied by afactor of 5 because of the dilution factor between the 0.5 ml of TSBcontaining the disk and the 100 μl that was plated onto the TSAII bloodagar plates.

Results

The silicone disk bioprosthetic colonization model has been previouslydescribed by Kuhn et al. (2002). This in vitro model is more clinicallyrelevant than the modified Robin device, in vitro model, in that itallows the silicone disk segments to be immersed in serum prior toexposing to high inoculum of bacteria or fungi. Furthermore, it allows ahigher concentration of adherence of bacteria and fungi on the siliconedisk of up to 5,000 CFU/disk (the modified Robbins device allows foronly 500 CFU/latex catheter segment). Because of the high inoculum thatthe silicone disk segments were exposed to in the bioprostheticcolonization model, the various disk segments were exposed to thevarious antimicrobial agents for one hour (rather than 15 minutes in themodified Robbins device). The results were consistent with the findingsand observations in the modified Robbins device model. As shown in Table4, exposure to either minocycline alone or EDTA or ethanol, or thecombination of minocycline and EDTA, failed to suppress thebioprosthetic MRSA colonization of the silicone disks. EDTA in 25%ethanol had some partial suppression but there was regrowth of theorganisms after 24 hour incubation. As expected, the control siliconedisk segments were heavily colonized before and after 24 hourreincubation. Minocycline in 25% ethanol was highly suppressive butthere was regrowth after 24 hour incubation. However, the triplecombination of M-EDTA in 25% ethanol was unique in completelyeradicating the MRSA organisms, with complete inhibition of regrowthafter 24 hours of incubation.

TABLE 4 Silicone Disk Bioprosthetic Colonization MRSA Model 24 h Growthafter 1 1 hour in Drug hour in Drug Drug Combinations Mean CFU ± std.Mean CFU ± std. Tested error error 3 mg/ml minocycline 5000.0 ± 0.0  5000.0 ± 0.0   n = 5 30 mg/ml EDTA 5000.0 ± 0.0   5000.0 ± 0.0   n = 525% EtOH in MHB 2900.0 ± 722.8  5000.0 ± 0.0   n = 10 3 mg/mlminocycline/30 mg/ml 5000.0 ± 0.0   3110.0 ± 637.6  EDTA n = 10 25%EtOH/30 mg/ml 730.0 ± 379.3 2770.0 ± 785.7  EDTA n = 10 25% EtOH/3 mg/ml0 85.0 ± 85.0 minocycline n = 10 25% EtOH/30 mg/ml 0 0 EDTA/3 mg/mlminocycline n = 10 Control (MH Broth alone) 5000.0 ± 0.0   5000.0 ±0.0   n = 10

Table 5 shows a similar trend for Candida parapsilosis. Minocyclinealone, EDTA alone, or the combination of M-EDTA failed to suppress oreradicate the growth of Candida parapsilosis on silicone disks.Furthermore, there was a heavy regrowth of the C. parapsilosis onsilicone disks after exposure to these agents and reincubation for 24hours. Twenty-five percent ethanol alone, EDTA in 25% ethanol orminocycline in 25% ethanol failed to completely suppress Candidaparapsilosis growth after one hour exposure and there was heavy regrowthafter 24 hour reincubation. The triple combination of M-EDTA in 25%ethanol completely eradicated the organisms on the silicone disks after1 hour exposure. Furthermore, the level of regrowth associated with atriple combination after 24 hours of reincubation was significantlylower than all the other alternative agents or their dual combination.

TABLE 5 Silicone Disk Bioprosthetic Colonization Candida ParapsilosisModel 24 h Growth after 1 1 hour in Drug hour in Drug Drug CombinationsMean CFU Mean CFU Tested C. Parapsilosis C. Parapsilosis 3 mg/mlminocycline 5000.0 ± 0.0   5000.0 ± 0.0   n = 5 30 mg/ml EDTA 5000.0 ±0.0   5000.0 ± 0.0   n = 5 25% EtOH in MHB  1933 ± 601.6 3875.0 ± 618.3 n = 10 3 mg/ml minocycline/30 mg/ml  4080 ± 585.0 500.0 ± 0.0  EDTA n =10 25% EtOH/30 mg/ml 1666.7 ± 629.9  2333.0 ± 666.7  EDTA n = 10 25%EtOH/3 mg/ml 1490.0 ± 542.4  5000.0 ± 0.0   minocycline n = 10 25%EtOH/30 mg/ml 0 582.9 ± 264.5 EDTA/3 mg/ml minocycline n = 10 Control(MH Broth alone) 5000.0 ± 0.0   5000.0 ± 0.0   n = 10

Thus, the two in vitro models of colonization (the modified Robbinsdevice as well as the silicone disk bioprosthetic colonization model)show that the triple combination is uniquely and highly effective ineradicating organisms embedded in biofilm on latex and silicone polymerswith minimal or no regrowth after 24 hour exposure to the combination.These two models are predictive of the clinical efficacy of this triplecombination in eradicating organisms embedded in biofilm on catheters ata temperature of 37° C.

Hence, the triple combination is superior in efficacy to the combinationof minocycline and EDTA, EDTA and ethanol or minocycline and ethanol.

All of the compositions and/or methods and/or apparati disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and/or apparati and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An antimicrobial solution comprising ethanol, EDTA and eitherminocycline or trimethoprim, wherein the concentration of ethanol is inthe range of 10% to 45% (v/v), the ethanol, EDTA and minocycline ortrimethoprim being present in amounts effective to eradicate Candidaparapsilosis or methicillin resistant S. aureas embedded in a biofilm.2. The solution of claim 1, wherein the concentration of the ethanol isin the range of 10-40%.
 3. The solution of claim 2, wherein theconcentration of ethanol is in the range of 15-30%.
 4. The solution ofclaim 3, wherein the concentration of the ethanol is about 25%.
 5. Thesolution of claim 1, comprising ethanol, minocycline and EDTA.
 6. Theantimicrobial solution of claim 1, wherein said solution is defined ascomprising ethanol, trimethoprim or minocycline, Ca(II)-EDTA, andpropylene glycol in a physiologically acceptable saline solution.
 7. Theantimicrobial solution of claim 1, wherein the solution comprisestrimethoprim.
 8. The antimicrobial solution of claim 7, furthercomprising propylene glycol.
 9. A syringe, comprising a unit dose of apharmacologically effective amount of a solution in accordance with anyone of claims 1, 2-4, 5, 7 or
 8. 10. A vial, comprising a unit dose of apharmacologically effective amount of a solution in accordance with anyone of claims 1, 2-4, 5 or 7-6.
 11. A medical device locking solutioncomprising a solution in accordance with any one of claims 1, 2-4, 5 or7.